Figure 5

Activation of UPR in Drosophila expressing the human N370S or L444P mutant variants. A. Protein lysates were prepared from 10 flies expressing the human UAS-mycHisWTGCase, human UAS-mycHisN370SGCase, or human UAS-mycHisL444PGCase, driven by Da^Gal4. Samples, containing 100 μg of protein, were subjected to overnight endo-H digestion, after which they were electrophoresed through SDS-PAGE and the corresponding blot was interacted with anti GCase and anti actin antibodies. Total GCase amount was divided by that of actin at the same lane and normalized to WT GCase, which was considered 100. The endo-H sensitive fraction before and after endo-H treatment, and the endo-H resistant fraction were labeled for convenience, as follows: endo-H sensitive before treatment: * [Endo-Hs(−)]; endo-H sensitive after treatment: # [Endo-Hs(+)]; endo-H resistant: • [Endo-Hr(+)]. B. RNA was isolated from the above-mentioned flies, and the cDNA prepared from it was subjected to quantitative RT-PCR with the appropriate primers. RP49 was used as a normalizing control. The results (three different experiments) were quantified as explained in the legend to Figure 3B and the values obtained for flies expressing normal human GCase were considered 1. Significance: * < 0.05; ** < 0.01. C. Protein lysates, prepared from the above–mentioned flies, were subjected to western blotting and interaction with anti phosphorylated eIF2α antibodies (p-eIF2α). As a loading control, the blot was interacted with anti eIF2α antibodies. D. The results (three different experiments) were quantified as explained in the legend to Figure 3B and the values obtained for wild type flies were considered 1. E. The number of larvae, (L3), that survived to the pupal stage, and the number of pupae that eclosed was counted. The number of larvae taken for each experiment was 90.